Streptococcus pneumoniae (Spn, the pneumococcus) remains a leading cause of respiratory tract and invasive infection. While vaccination with capsular polysaccharide-based vaccines has decreased the high burden of Spn disease, this approach fails to target the majority of Spn, which are non-vaccine serotypes. Unfortunately, the development of more broadly-acting, serotype- independent vaccines that focus on preventing Spn disease has proven to be elusive. The required first step for Spn-host interaction, however, is colonization of the mucosal surfaces of the upper airways. Our premise is that interrupting colonization will have the greatest overall impact on Spn disease and offers new, unexplored possibilities for prevention. We have optimized an infant mouse model of Spn colonization that has allowed the application of Tn-Seq transposon mutagenesis to interrogate the entire Spn genome to identify loci affecting colonization. The complete dataset of non-essential Spn genes affecting colonization was then compared to bacterial surface factors that are immunogenic during human infection. Defined mutants in each of these candidates were constructed and tested in both infant and adult murine models of colonization. Seven loci expressing proteins of known function that modify the bacterial surface or host substrates were validated as required for efficient colonization. These proteins are all members of the Spn core genome present in all strains and show minimal sequence variation across isolates. Our hypothesis is that immunity to these candidates, alone or in combination, will interrupt Spn colonization. This hypothesis will be tested first by immunization with recombinant protein in adjuvant (SC or IN) to maximize antibody responses. Murine antibody will be used to confirm surface localization and to explore its ability to block protein function using in vitro assays. Immune mice v. adjuvant alone controls will then be challenged IN or via pup-to-pup transmission to assess protection from colonization with diverse strains. In future studies, candidates validated in the murine model can be investigated using experimental human pneumococcal carriage to bridge the critical step from animal to human testing. Thus, our proposal takes a novel approach, targeting colonization, to the ongoing public health problem of the pneumococcus.